Western lifestyle and diet have spurred the growth of numerous obesity related disorders in the US, including metabolic syndrome and type 2 diabetes, to epidemic proportions. However, less well known is the rising epidemic of non-alcoholic fatty liver disease (NAFLD). NAFLD ranges from mild hepatosteatosis to severe fibrosis or cirrhosis and is initially characterized by abnormal accumulation of lipids in the live. The medical significance of NAFLD is reflected in its sheer numbers; NAFLD is the single largest cause of abnormal liver blood tests in the clinic and is the most common chronic liver disease in adults and children. In addition, the majority of type 2 diabetics have some form of NAFLD. Despite the severity of the problem, there is no FDA- approved small molecule drug treatment for NAFLD. Our proposal seeks to translate discovery of a set of promising small molecules that treat the underlying cause of lipid accumulation in an animal model of NAFLD. We propose to further develop these small molecules to advance optimized leads toward the clinic. To achieve this goal, we have established a phenotypic drug discovery platform for fatty liver disease incorporating in vitro and in vivo models. In Phase I, we developed novel small molecules that modulate lipid metabolism and stimulate lipid clearance in lipid-loaded human cells, and demonstrate in vivo efficacy. This Phase II project will develop a series of compounds based on leads with proven in vivo therapeutic value towards increasing efficacy, limiting toxicity and identifying new biomarkers, with the longer-term goal of generating preclinical IND-enabling efficacy and ADME/Tox data. Aim 1 will optimize small molecule efficacy through medicinal chemistry/SAR. Analogs (~200) will be screened in vitro using a suite of metabolically significant protein and mRNA biomarkers (including FGF21, Car3). Aim 2 will evaluate the screened molecules (~50) for efficacy in vivo in the C57-DIO mouse model of NAFLD. The extent of hepatic lipid accumulation/clearing will be measured using conventional triglyceride assay kits and histological scoring. Compounds exhibiting increased efficacy/potency will feed back into Aim 1 to guide further compound derivative synthesis and testing. Aim 3 will establish therapeutic potential and toxicity/metabolic profiling in primary human hepatocytes with evaluation in an ex vivo fatty liver model. Up to 10 molecules will be assessed using primary human hepatocytes, and 2-3 molecules will be evaluated in an ex vivo perfusion model for fatty liver. The output of this aim is to select a preclinical candidate for advancement towards full ADME/Tox studies likely in Phase IIB.